Project Summary Alcohol use disorder (AUD) is a chronic, debilitating, and relapsing brain disease. An overwhelming number of people die yearly due to alcohol-related causes; however, the mechanism(s) of action are challenging to decipher and remain largely elusive. Long noncoding RNAs (lncRNAs) are key regulators of the genome; persistent transcriptional changes induced by chronic ethanol exposure is a hypothesized mechanism for AUD development, withdrawal, and relapse. Our laboratory and others have shown that ethanol-responsive lncRNAs have the ability to directly regulate ethanol- related behavior when mutated in vivo, but only a handful have been characterized to date. In this proposal, I will learn and apply state-of-the-art CRISPR/Cas9 techniques to functionally investigate ethanol-responsive lncRNA Gas5 for its regulation of ethanol drinking and ethanol-related behaviors. As ethanol alters gene expression and molecular pathways that regulate neuroinflammation, I want to target an ethanol-responsive and highly-interconnected competing endogenous RNA (ceRNA) lncRNA that may coordinate large endogenous immune networks related to AUD. I will focus on in vivo CRISPR/Cas9 modulation of a specific ‘hub’ lncRNA of interest, growth arrest-specific 5 (Gas5). Gas5 acts as a ceRNA to regulate immune signaling, is significantly and persistently downregulated following chronic intermittent ethanol vapor exposure and has been shown to reduce cocaine-intake, linking Gas5 to substance abuse. It is therefore of substantial interest to test the hypothesis that Gas5 is a key determinant of ethanol action. Gas5 will be knocked out in a temporal, cellular, and brain region-specific manner to investigate its role on ethanol drinking. Upon completion of this project, I will have advanced our understanding for the molecular impact of ethanol- responsive lncRNA Gas5 and how it relates to addictive behavior. This project will provide substantial training opportunities, a strong research foundation applicable to a variety of scientific fields, and a collaborative environment with other researchers in the field including the Integrative Neuroscience Initiative on Alcoholism – Neuroimmune consortium. A wealth of technical skills ranging from bench work to complex surgical techniques, the understanding and development of project design, and several scientific writing manuscript prospects are available within each aim that will be valuable for my continued scientific training.